**2. Two phases of GSIS**

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

a closure of the ATP-sensitive K<sup>+</sup>

glucose intake contributes to the increasing pentose phosphate pathway (PPP) supply of NADPH for NADPH oxidase 4 (NOX4), which directly produces H2O2. The burst of H2O2 then represents a redox signal, which fundamentally determines GSIS, while inducing a cooperative induction of plasma membrane depolarization together with ATP elevation (**Figure 1**) [1]. The latter originates from the increased ATP synthesis by oxidative phosphorylation (OXPHOS). Hypothetically, either

ATP; or H2O2 activates a synergic channel such as transient receptor potential

*(A) Traditional ("standard") view of the triggering mechanism of GSIS compared with (B) new paradigm in GSIS mechanism ("novel"), for which the redox signaling by NOX4-produced H2O2 is essentially required. Upon the glucose intake, PPP and redox shuttles supply cytosolic NADPH to increase NOX4 activity and* 

*exclusively when both ATP plus H2O2 are elevated. Alternatively, H2O2 activates opening of TRPM2 or other nonspecific cation channels required for a depolarization shift to reach a threshold potential of* −*50 mV, at which the voltage-sensitive Ca2+ channels (CaL) become open, thus starting to fire the action potential. Resulting Ca2+ influx into the cell cytosol allows a complex process of exocytosis of the insulin granule vesicles (IGVs), beginning during the so-called 1st phase of GSIS by fusion of pre-attached IGVs with the plasma membrane and exposure of the IGV interior to the extracellular space (capillaries in vivo). Ca2+ also promotes the recruitment of distant IGVs towards the plasma membrane as well as ensures the late, so-called 2nd phase* 

 *channel (KATP) is closed* 

*thus elevate H2O2 which substantiates redox signaling. Either, the ATP-sensitive K+*

channel (KATP) is dependent on both H2O2 plus

**34**

*of GSIS, lasting about 1 hour in vivo.*

**Figure 1.**

Two phases exist for GSIS in vivo [7–11]. They are also recognized in isolated pancreatic islets (PIs), but not in insulin-secreting β-cell lines. The consensus became that both KATP-dependent mechanism (also termed "triggering") and KATP-independent mechanisms contribute to both phases [12], while the KATPindependent mechanism still requires the elevation of cytosolic Ca2+ [13]. The 2nd phase in vivo was even considered to be independent of the extracellular glucose concentrations [14]. It depends more on the molecular mechanism of the increased sustained mobilization and priming of insulin granule vesicles (IGVs) [15].

The first rapid peak of insulin secretion is observed at 5–10 min after administration of a bolus of glucose in vivo or addition of glucose to the isolated PIs. The 1st phase involves the exocytosis of pre-docked juxtaposed IGVs, residing 100–200 nm from the plasma membrane prior to triggering [16, 17] and also possesses a contribution of deeper localized granules arriving within 50 ms, which were not initially pre-docked [18, 19]. The 2nd phase typically lasts over 1 hr. As a result, a predominant insulin amount is released in this phase. The 2nd phase results most likely from further delayed recruitment of IGVs belonging to the typically excessive reserve. The past hypothesis suggested a main reason for such a delay to involve the restricted passage through the filamentous actin (F-actin) cytoskeleton [20–22], but later a microfilament-independent movement of IGVs was reported [21, 23–25]. However, numerous cytoskeleton components play a more detailed role in the IGV exocytosis, not representing only a simple barrier. Generally, the IGV exocytosis relies on synaptogamin activation by Ca2+, syntaxin, SNAP-25, and other target proteins of the SNARE family (SNAp REceptors, where SNAP is soluble NSF attachment proteins and NSF is a N-ethylmaleimide-sensitive fusion factor). They attract IGVs via the IGV-localized synaptobrevins (vesicle-associated membrane proteins), while forming a coiled-coil quarternaly structure [26]. The resulting SNARE core complex relocates the IGV and plasma membrane into proximity, thus facilitating establishment of so-called fusion stalk. Further zippering of coiled-coil structures allows fusion of larger part of the IGV membrane with the plasma membrane until a fusion pore is formed.

However, the recent explanation for the second phase is based on the fact that the two phases of insulin secretion exist when isolated pancreatic islets are studied, but do not exist for isolated primary pancreatic β-cells [27–29]. Hence, the role of inter-cellular contacts is emphasized for the 2nd phase. The inter-cellular contacts allow synchronization of the plasma membrane potential, while paracrine hormone secretion may also contribute to modification and termination of insulin release.
